Integrative Physiology Cardiac Myocyte KLF5 Regulates Ppara Expression and Cardiac Function Konstantinos Drosatos,* Nina M. Pollak,* Christine J. Pol, Panagiotis Ntziachristos, Florian Willecke, Mesele-Christina Valenti, Chad M. Trent, Yunying Hu, Shaodong Guo, Iannis Aifantis, Ira J. Goldberg
Rationale: Fatty acid oxidation is transcriptionally regulated by peroxisome proliferator–activated receptor
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(PPAR)α and under normal conditions accounts for 70% of cardiac ATP content. Reduced Ppara expression during sepsis and heart failure leads to reduced fatty acid oxidation and myocardial energy deficiency. Many of the transcriptional regulators of Ppara are unknown. Objective: To determine the role of Krüppel-like factor 5 (KLF5) in transcriptional regulation of Ppara. Methods and Results: We discovered that KLF5 activates Ppara gene expression via direct promoter binding. This is blocked in hearts of septic mice by c-Jun, which binds an overlapping site on the Ppara promoter and reduces transcription. We generated cardiac myocyte–specific Klf5 knockout mice that showed reduced expression of cardiac Ppara and its downstream fatty acid metabolism–related targets. These changes were associated with reduced cardiac fatty acid oxidation, ATP levels, increased triglyceride accumulation, and cardiac dysfunction. Diabetic mice showed parallel changes in cardiac Klf5 and Ppara expression levels. Conclusions: Cardiac myocyte KLF5 is a transcriptional regulator of Ppara and cardiac energetics. (Circ Res. 2016;118:241-253. DOI: 10.1161/CIRCRESAHA.115.306383.) Key Words: cardiac myocyte
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atty acid oxidation (FAO) accounts for the production of ≈70% of the ATP that the heart uses.1 Some forms of heart failure are due to perturbations in heart energetics, and severe heart failure is associated with energy starvation and reprogramming of cardiac energetics.2 These metabolic changes occur regardless of whether the primary cause of cardiac dysfunction is metabolic disease, pressure overload, or ischemia.3,4 A dramatic example of cardiac dysfunction due to reduction in FAO5 and energy depletion occurs in sepsis.5–7 The transcriptional mechanisms that underlie inhibition of cardiac FAO and cardiac dysfunction during sepsis and other types of cardiac dysfunction are incompletely understood. Cardiac FAO is regulated at several stages: FA uptake, triglyceride formation and storage in lipid droplets, triglyceride lipolysis leading to release of unesterified fatty acids, and transfer of fatty acids into the mitochondria for FAO and ATP production. Most of the proteins that participate in this cascade are transcriptionally regulated by peroxisome proliferator–activated receptor α (PPARα).8 Although it is known
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PPAR alpha
that PPARα is activated by FAs that are released via lipolysis from the intracellular triglyceride pool,9,10 the transcriptional regulation of Ppara is not fully elucidated. Various gain or loss of PPARα function animal models resulted in mixed outcome with either protective or aggravating roles of PPARα in cardiac function. Α variety of metabolic and pathological stress conditions influence cardiac PPARα expression in multiple ways, which are not fully defined.
Editorial, see p 193 Metabolism in several tissues is regulated by members of the Krüppel-like factor (KLF) protein family, which regulate proliferation, differentiation, development, and cell death.11 Thus far, 17 KLF isoforms have been identified in humans and mice, whereas several homologs were described in other species.11 Adipocyte KLF2,12 KLF3,13 and KLF714 inhibit adipose tissue development. However, KLF4,15 KLF6,16 and KLF1517 have the opposite effect in adipocytes because they induce Pparg and adipogenesis. Hepatic KLF11 induces Ppara and
Original received March 4, 2015; revision received November 12, 2015; accepted November 16, 2015. In October 2015, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15.18 days. From the Metabolic Biology Laboratory, Department of Pharmacology, Center for Translational Medicine, Temple University School of Medicine, Philadelphia, PA (K.D., C.J.P., M.-C.V.); Institute of Molecular Biosciences, University of Graz, Graz, Austria (N.M.P.); Howard Hughes Medical Institute, Department of Pathology, New York University School of Medicine (P.N., I.A.); Division of Endocrinology, Diabetes, and Metabolism, New York University-Langone School of Medicine (F.W., C.M.T., Y.H., I.J.G.); and Division of Molecular Cardiology, Department of Medicine, Texas A & M Health Science Center, Temple (S.G.). *These authors contributed equally to this article. The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA. 115.306383/-/DC1. Correspondence to Konstantinos Drosatos, PhD, Metabolic Biology Laboratory, Department of Pharmacology, Temple University School of Medicine, Center for Translational Medicine, 3500 N. Broad St, Philadelphia, PA 19140. E-mail
[email protected] © 2015 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.115.306383
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242 Circulation Research January 22, 2016
Nonstandard Abbreviations and Acronyms FAO FA PPARα KLF SGLT αMHC
fatty acid oxidation fatty acid peroxisome proliferator–activated receptor α Krüppel-like factor sodium/glucose cotransporter α myosin heavy chain
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FAO genes and prevents hepatic triglyceride accumulation.18 KLF15 promotes lipid use in the heart19 and skeletal muscle.20 Thus, several KLF isoforms have been implicated in the regulation of metabolic pathways in several organs, including the heart. KLF5 is involved in pressure overload–mediated cardiac hypertrophy, but its role in cardiac metabolism remains unknown. Heterozygote Klf5+/− mice are protected from pressure overload cardiac hypertrophy21 because of reduced transforming growth factor β production in cardiac fibroblasts and not because of changes in cardiac myocytes.22 Heterozygote Klf5+/− mice showed increased skeletal muscle FA consumption because of activation of PPARδ,23 suggesting that KLF5 is an inhibitor of lipid catabolism. Conversely, Klf5 deletion inhibited lipid production in lung surfactant,24 indicating that KLF5 is a positive regulator of lipid homeostasis in lungs. Thus, the actions of KLF5 in lipid metabolism vary depending on its site of expression. We focused on the role of KLF5 in the regulation of cardiac metabolic gene expression. Unexpectedly, we first discovered that Klf5 gene expression was induced in energy-depleted hearts of mice treated with Escherichia coli lipopolysaccharides that had lower Ppara expression. Although this observation implicated cardiac KLF5 in Ppara and FAO inhibition, our subsequent studies showed the opposite. We created a cardiac myocyte–specific Klf5−/− mouse and conducted gainof-function experiments in cardiac myocytes that revealed KLF5 to be a transcriptional activator of Ppara. Klf5 ablation in cardiac myocytes reduced cardiac FAO and ATP content, increased triglyceride accumulation and caused cardiac dysfunction. Furthermore, cardiac KLF5 was reduced in the early stages of type 1 and in type 2 diabetes mellitus mouse models along with Ppara gene expression. Thus, KLF5 is a novel regulator of Ppara and cardiac lipid use.
Methods
Expanded Methods are presented in the Online Data Supplement. All animal studies were approved by the institutional animal care and use committees. Data are expressed as the mean±SEM. Statistical significance was assessed with t test or 1-way ANOVA followed by Bonferroni post hoc tests, performing all pairwise comparisons. A P
